Recombinant Arabidopsis thaliana Oleosin 18.5 kDa (At4g25140)

What is the biological function of Oleosin 18.5 kDa in Arabidopsis thaliana?

Oleosin 18.5 kDa (OLEO1) is the most abundant oil body-associated protein in Arabidopsis thaliana seeds, accounting for approximately 65% of all oil body proteins . It serves several critical functions:

• Oil body formation and stabilization: OLEO1 prevents oil body coalescence during seed desiccation and storage by creating a steric hindrance and electrostatic repulsion between oil bodies .

• Triacylglyceride (TAG) solubilization: It helps maintain TAGs in a fluid state within the hydrophobic environment of the seed cell .

• Germination support: During germination, OLEO1 facilitates controlled TAG mobilization for energy provision to the growing seedling .

Research using RNA interference to suppress OLEO1 expression has demonstrated that seeds lacking this protein develop abnormally large oil bodies, confirming its role in determining oil body size and stability .

How should recombinant Arabidopsis thaliana Oleosin 18.5 kDa be stored and reconstituted for experimental use?

Storage Protocol:

• Store lyophilized powder at -20°C to -80°C upon receipt

• Avoid repeated freeze-thaw cycles

• Working aliquots may be stored at 4°C for up to one week

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard)

• Aliquot for long-term storage at -20°C to -80°C

The protein is typically provided in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 . For applications requiring different buffer conditions, performing a buffer exchange via dialysis is recommended to maintain protein stability.

What experimental approaches can be used to study Oleosin 18.5 kDa expression and localization?

Several methodological approaches are available for studying Oleosin 18.5 kDa expression and localization:

Expression Analysis:

• Western Blotting: Using specific antibodies against Oleosin 18.5 kDa or the His-tag (for recombinant proteins). SDS-PAGE separation followed by immunoblotting can quantify expression levels .

• RT-qPCR: For transcriptional analysis of the At4g25140 gene across different developmental stages or in response to experimental treatments.

Localization Studies:

• Subcellular Fractionation: Isolating oil bodies through flotation centrifugation followed by protein extraction.

• Immunofluorescence Microscopy: Using fluorescently-labeled antibodies against Oleosin 18.5 kDa to visualize its localization on oil bodies.

• Fluorescent Protein Fusions: Creating GFP or YFP fusions with Oleosin 18.5 kDa for live-cell imaging of oil body dynamics.

For optimal results, researchers should use fresh samples when possible, as Oleosin properties can change during storage due to oxidation of the protein's hydrophobic domain .

How does suppression or overexpression of Oleosin 18.5 kDa affect oil body morphology and seed physiology?

Genetic manipulation of Oleosin 18.5 kDa expression produces significant phenotypic effects with implications for both basic research and biotechnological applications:

OLEO1 Suppression Effects:

• Oil Body Morphology: RNA interference-mediated suppression of OLEO1 results in dramatically enlarged oil bodies within seed cells, demonstrating that oleosin abundance inversely correlates with oil body size .

• Germination Efficiency: OLEO1-suppressed lines show altered germination kinetics, suggesting that proper oil body structure affects the accessibility of lipases to stored TAGs during germination .

• TAG Mobilization: Reduced OLEO1 levels affect the rate and efficiency of TAG mobilization during germination, with potential changes in fatty acid release patterns .

OLEO1 Overexpression Effects:

• Oil Body Stability: Increased OLEO1 expression can lead to smaller, more stable oil bodies.

• Phenotype Rescue: Recombinant oleosin expression can reverse the large oil body phenotype in OLEO1-suppressed lines, confirming the direct relationship between oleosin abundance and oil body size .

Methodology for studying these effects includes:

• Transmission electron microscopy of seed cells

• Quantitative image analysis of oil body size distribution

• Germination assays under various conditions

• Gas chromatography analysis of fatty acid profiles during germination

What is the relationship between Oleosin 18.5 kDa and other oil body proteins in Arabidopsis thaliana?

Arabidopsis thaliana oil bodies contain a complex proteome dominated by four oleosin isoforms that interact functionally:

Additional oil body-associated proteins include:

• Caleosin: Contains calcium-binding motifs

• Steroleosin: Has sterol-binding and dehydrogenase domains

These proteins exhibit functional relationships with important research implications:

• Compensatory Mechanisms: Suppression of OLEO1 does not trigger significant upregulation of other oleosin isoforms, suggesting limited functional redundancy .

• Developmental Regulation: Different isoforms show distinct temporal expression patterns during seed development.

• Degradation Pathways: Higher molecular weight isoforms (OLEO2, OLEO4) are preferentially targeted for ubiquitination at their C-terminal regions during germination, suggesting differential regulation of oil body protein turnover .

Methodological approaches for studying these relationships include:

• Comparative proteomics of oil bodies across development

• Co-immunoprecipitation to identify protein-protein interactions

• Multiple gene knockout/knockdown studies to assess combinatorial effects

How can the Oleosin 18.5 kDa fusion system be optimized for recombinant protein expression?

The oleosin fusion technology represents a powerful platform for recombinant protein expression in plants, offering unique advantages for protein production and purification. Optimization involves several methodological considerations:

Fusion Design Parameters:

• Fusion Position: C-terminal fusions to Oleosin 18.5 kDa have proven most successful, preserving oil body targeting while allowing the fusion partner to extend into the cytosol .

• Linker Selection: Incorporating flexible linkers (glycine-serine repeats) between Oleosin 18.5 kDa and the target protein improves folding and accessibility of the fusion partner.

• Codon Optimization: Plant bias codon usage-optimization significantly enhances expression levels, as demonstrated with human insulin-like growth factor 1 (hIGF-1) fusions .

Expression Optimization:
The oleosin promoter driving the fusion gene expression has demonstrated high efficiency in Arabidopsis seeds. Using this system, oleosin-hIGF-1 fusion protein accumulated to 0.75% of total seed protein, with the hIGF-1 portion representing 0.17% - eight times higher than other plant-based hIGF-1 production systems .

Purification Strategies:

• Oil body isolation via flotation centrifugation

• Target protein release through protease cleavage at engineered sites

• Further purification using affinity chromatography

This technology has been validated with bioactive human insulin-like growth factor 1, demonstrating that the fusion protein retained biological activity in vitro when tested on human SH-SY5Y neuroblastoma cells .

What mechanisms regulate Oleosin 18.5 kDa degradation during seed germination?

During germination, Arabidopsis thaliana seeds undergo a coordinated process of oil body breakdown to utilize stored TAGs as an energy source. This process involves regulated degradation of Oleosin 18.5 kDa through specific mechanisms:

Ubiquitination Pathway:

• Recognition: Specific E3 ligases recognize and target oleosins for ubiquitination.

• Site Specificity: While higher molecular weight oleosins are predominantly ubiquitinated at their C-terminal regions, the specific ubiquitination sites on Oleosin 18.5 kDa require further characterization .

• Proteasomal Degradation: Ubiquitinated oleosins are targeted to the 26S proteasome for degradation, facilitating the exposure of TAG surfaces to lipases.

Temporal Regulation:
Oleosin degradation occurs in a precise temporal sequence, with different isoforms showing distinct degradation kinetics. This sequential breakdown coordinates with lipase recruitment and TAG hydrolysis .

Methodological Approaches:

• Proteomic Analysis: Mass spectrometry analysis of oleosins isolated from seeds during germination to identify ubiquitination sites.

• Immunological Detection: Dual antibody approach against both oleosin and ubiquitin to track modification status during germination .

• Inhibitor Studies: Using proteasome inhibitors or deubiquitinating enzyme inhibitors to determine pathway dependencies.

• Mutant Analysis: Studying germination in Arabidopsis lines with mutations in specific E3 ligases or proteasome components.

Understanding these degradation mechanisms has significant implications for both basic seed biology and biotechnological applications aimed at modifying oil mobilization during germination.

How does the amino acid sequence of Oleosin 18.5 kDa contribute to its targeting and integration into oil bodies?

The unique structural features of Oleosin 18.5 kDa enable its specific targeting and integration into oil bodies through a precisely orchestrated process:

Domain-Specific Functions:

• N-terminal Hydrophilic Domain (Amino acids 2-41): Contains signals for endoplasmic reticulum targeting and may interact with phospholipids at the oil body surface.

• Central Hydrophobic Domain (Amino acids 42-117): Forms a hairpin structure that penetrates into the triacylglyceride core of the oil body. This domain contains a distinctive proline knot motif (PX5SPX3P) that facilitates the hairpin fold .

• C-terminal Hydrophilic Domain (Amino acids 118-173): Remains exposed on the oil body surface and can serve as an attachment site for fusion proteins. This domain contains lysine residues that may serve as ubiquitination sites during germination .

Targeting Mechanism:
The oleosin targeting pathway involves co-translational integration into the endoplasmic reticulum membrane. The signal recognition particle binds to a signal region within the developing polypeptide chain of the oleosin/ribosome complex, targeting it to the endoplasmic reticulum where synthesis completes with the protein embedded in the membrane .

Experimental approaches to study targeting include:

• Site-directed mutagenesis of specific domains

• Creation of chimeric proteins with domain swaps

• Live-cell imaging of fluorescently-tagged oleosin variants

• In vitro reconstitution systems using artificial oil bodies

What experimental considerations are important when designing studies involving Recombinant Arabidopsis thaliana Oleosin 18.5 kDa?

Designing robust experiments with Recombinant Arabidopsis thaliana Oleosin 18.5 kDa requires attention to several critical factors:

Protein Quality Control:

• Purity Assessment: Ensure protein purity >90% via SDS-PAGE before experimental use .

• Activity Verification: For fusion proteins, verify biological activity of the fusion partner using appropriate bioassays, as demonstrated with oleosin-hIGF-1 fusions and human SH-SY5Y neuroblastoma cells .

• Aggregation Monitoring: Check for protein aggregation using dynamic light scattering or size-exclusion chromatography, particularly after reconstitution from lyophilized form.

Experimental Design Considerations:

• Controls: Include appropriate controls for both N-terminal His-tagged oleosin and untagged versions to account for tag effects on protein behavior.

• Concentration Determination: Accurately determine protein concentration using methods that account for the high hydrophobicity of oleosins.

• Buffer Compatibility: Test compatibility with experimental buffers, as the hydrophobic domain can cause unpredictable behavior in different buffer systems.

• Storage Effects: Consider potential oxidative modifications during storage, particularly to the hydrophobic domain, which may affect functional properties.

In vivo vs. In vitro Studies:
When comparing recombinant protein behavior with native oleosins, consider:

• Post-translational modifications present in plant-derived oleosins but potentially absent in E. coli-expressed proteins

• Potential folding differences between eukaryotic and prokaryotic expression systems

• Differences in lipid environments between artificial and natural oil bodies

For genetic studies using Arabidopsis, the SALK_072403 line provides a valuable oleosin knockout resource that can be complemented with various oleosin constructs to test structure-function hypotheses .